The overall goal of this research is to chemical and structural motifs linked to the mechanism of action of membrane-interacting antimicrobial peptides. The increased prevalence of antibiotic resistance in numerous bacterial strains has a tremendous annual impact, especially in hospital settings for patients with compromised immune systems. As more strains develop resistance to more antibiotics it underscores the need for development of new antimicrobials that have a low propensity for resistance development. One potential source of such antimicrobials are naturally occurring host-defense peptides (HDPs) which have been found in a variety of organisms throughout the evolutionary chain, indicative that bacteria have not been able to mount a sufficient resistance response to these molecules. The HDPs are typically short peptides (10-50aa) that bind to bacterial cell surfaces with high affinity and high selectivity over host cells. The mechanism of action of the peptides is still a matter of debate, but most HDPs exhibit some degree of membrane destabilization upon binding to the lipid bilayer which is likely linked to the mechanism of action. Recent studies have also shown a number of bacterial transcriptional responses induced by exposure to HDPs, many of which are not completely understood. Mechanism of action studies are further complicated by the sheer number of HDPs isolated from natural sources, many of which may use somewhat different mechanisms based on the host and what pathogens the host routinely encounters. The goal of the proposed research is to focus on the mechanism of action of several naturally derived alpha- helical HDPs selected for diverse host organisms and broad spectrum antimicrobial activity. A deeper understanding of the mechanism of alpha-helical HDPs relies on understanding the structure-activity relationship in these peptides. Furthermore, as relatively small peptides, the HDPs are experimentally tractable and valuable biophysical model for understanding protein-membrane interaction and as a model system for exploring the principles of membrane protein folding. Our strategy for studying the behavior of HDPs involves the synthesis of peptides with natural and altered peptide sequences designed to examine the role(s) of specific chemical moieties in the activity of the peptide. This will be achieved by rationally incorporating either naturally occurring or synthetic amino acids to replace similar residues at a series of sites within the peptide, monitoring the binding of modified peptides to lipid bilayers, and testing the antimicrobial activity of the newly designed peptides. Multiple types of optical spectroscopy will be employed to characterize the membrane binding and structural transitions in the peptides. Fluorescence emission spectroscopy is a sensitive probe of environmental changes (i.e. aqueous peptide binding to a less polar lipid membrane environment) while numerous fluorescence quenching assays can determine the depth of penetration of the peptide/fluorophore in the bilayer. Circular dichroism and Forster resonance energy transfer studies will elucidate peptide folding at the bilayer surface. Finally absorbance and fluorescence spectroscopy will be used in the bacterial membrane permeabilization assays, one of the proposed mechanisms of action of HDPs. The naturally occurring HDPs will first be examined with no sequence alterations and rationally designed analogues will be synthesized after this initial characterization. Sequence modifications focusing on the cationic amino acids in the sequence as well as modification of net charge/hydrophobicity of the HDPs will be the central experiments. HDP binding affinity and secondary structure data will be collected for a variety of lipid bilayer and detergent micelle compositions to determine lipid-based effects on HDP structure and mechanism of action. Other environmental factors that may impact mechanism of action including ionic strength and pH will also be investigated. Antimicrobial activity and bacterial membrane permeability assays will be performed using both gram positive and gram negative strains of bacteria. New, more broadly applicable assays for membrane permeabilization will also be developed. PUBLIC HEALTH RELEVANCE: This project involves determining the mechanism of action of a number of naturally occurring host defense peptides (HDPs), which are an essential component of the innate immune system in most higher organisms. The development of resistance to small-molecule and synthetic antimicrobials is a major public health concern, both in its annual death toll and its potential to develop "superbugs" or bacterial strains which are resistant to multiple last-line antibiotics. A full understanding of the structure-activity relationship in the mechanism of action of HDPs from varied host organisms is a key component necessary for the rational design of new antimicrobials with low potential for resistance development.